This invention relates to transformation of plants via the pollen tube pathway through applications of DNA solutions directly to the surface remaining after styles of florets are removed following pollination. In another aspect, the direct transformation of plants is achieved by applying solutions containing donor DNA directly to a wound site on the ovary following removal of styles which has been previously pollinated with mentor pollen while allowing for the formation of pollen tubes extending from the style into the ovule.
Plant transformation has been achieved through various means. The first to be developed and most frequently used employs Agrobacterium tumefaciens as a vector to introduce alien DNA into the genome of the targeted plant. This technique was initially limited to the host plants of A. tumefaciens (i.e. dicotyledons), but was recently successfully applied to a monocot, corn, Zea mays L., [Gould et al, Plant Physiol., 95, 426-434 (1991)]. However, success of the technique remains dependent upon the ability to regenerate plants from infected explants. For most important crops, this ability has not been developed or is not compatible with the needs of the transformation technique through A. tumefaciens. 
Other currently known techniques for transformation, include electroporation and bombardment with DNA-coated microprojectiles. In the case of electroporation, the technique has only been successful for species that can be regenerated from protoplasts.
Microprojectile bombardment to introduce alien DNA into the genome of a host plant is exemplified. In soybean, Glycine max L., bombardment of over 20,000 shoot-apices only resulted in transient expression of the transforming gene whereas bombardment of embryogenic suspensions with the same gene produced several transformed plants.
Current techniques of plant transformation, i.e. electroporation and microprojectile bombardment require the ability to regenerate plants from single cells. For most crops, this ability has yet to be developed despite repeated efforts. These techniques also require sophisticated technical skills and facilities.
Accordingly, there is a need for a technique of direct plant transformation with fewer requirements that will be applicable to most crops, easy to implement and cost effective. There is a need for a technique of direct transformation that eliminates the need to regenerate plants from cultured explants.
The delivery of foreign DNA into plants via pollen tube pathway has been reported. In earlier work, the total genomic DNA from a donor plant were injected into the axial placenta of recipient plants about a day after self-pollination. The genomic DNA was partially protected from shearing and hydrolysis by partially recombination of the DNA with histones. It was found that foreign DNA could transform the embryo by entering the ovule, following the path along with the pollen tube grew, and a great number of mutant offsprings were obtained. However, no molecular evidence was presented to confirm that the phenotypic changes seen in the mutant offsprings of recipient plants were caused by the exogenous DNA from the donor plants.
The present invention relates to a method for transformation of plants by direct application of exogenous DNA solution into a wound site on the ovary after removal of styles which have been previously pollinated with mentor pollen, allowing for the formation of pollen tubes extending from the style into the ovule. Preferably, the exogenous DNA sequence will correspond to a desired physical or functional characteristic to be imparted to the host plant. The DNA sequence is introduced into the host plant genome, thus producing a transformed plant embryo.
Current methods of transformation generally require passage through a tissue culture cycle, thus, only plants amenable to culture (i.e. regeneration capability) are used as recipients. Few culture systems have been worked out; however, the present inventive method is attractive for the transformation of many different plants because there is no need for tissue culture. The present methodology does not require the bombardment of tissues, and unlike these prior taught systems, does not require expensive equipment and personnel trained in the art of tissue culture. The present method is relatively simple and it can be used to introduce multiple gene constructs in a single application, unlike other systems. The results achievable are like a backcross method of breeding plants. The direct introduction of DNA without markers would be more stably incorporated into genomes.
Even though the direct transformation of higher plants through pollen tube pathway is suitable for a variety of flowering plants, the present disclosure is focused on the onion plant. Onion is an important monocotyledonous vegetable grown on all the continents of the world. The common bulb-type onion, Allium cepa L., are by far the most important onions in commercial trade. Depending on its adaptation, it can bulb under either long-or short-day conditions. It produces a single bulb and has an umbel type inflorescence that produces true seeds. Bulbing is controlled by a combination of day length and temperature. Several closely related species include A. cepa, including the edible species A. fistulosum L. (Japanese bunching or Welsh onion), A. satiuum L. (garlic), A. ampeloprasum L. (great-headed garlic, leek, and Kurrat), A. chinene Maxim (rakkyo), A. schoenoprasum L. (chives) and A. tuberosum L. (Chinese chives). Onion has become an essential part of man""s diet mainly for its flavoring qualities. There seems to be no limit to its use by any nationality.
In the United States, onions are produced in many regions as one of the major vegetables. In 1992, it ranked third in both acreage and value among the eleven principal commercial vegetables with a total of 147,200 acres and $613,620,000. In Texas, onion was the number one vegetable crop with 19,500 acres of production and had a value of $61,022,000 in 1992 (USDA, Vegetables Annual Summary, 1992).
Onion has been greatly improved in characteristics such as quality, yield, and uniformity by classic plant breeding methodology. Development of onion cultivars resistant to fungal, bacterial, and viral diseases has been a major project by public and private breeders for many years. However, genetic variability for disease resistance within accessions of A. cepa is limited, but resistance to some of the diseases has developed within locally adapted A. cepa forms. Some related species (A. fistulosum, A. galanthum, A. royelii) could contribute germplasm for resistances in breeding programs. Unfortunately, resistance from these related species have not successfully been introduced into commercial A. cepa cultivars because of post-fertilization barriers such as hybrid embryo abortions during early embryo development in interspecific hybridization in the genus Allium. Ovule culture was not sufficient to overcome post-fertilization barriers restraining normal hybrid plant development (Bino et al., 1989; Gonzalez and Ford-Loyd, 1987). Besides a high degree of hybrid sterility, limited genetic recombination between the different genomes of Allium also limits the use of interspecific hybridization for onion improvement (Novak et al., 1986). One of the ways to bypass the pre- or post-fertilization barriers in interspecific hybridization is genetic transformation.
Genetic transformation transcends classical plant breeding by permitting the rapid transfer of genetic traits between entirely different organisms. Potential benefits include enhanced nutritional value from crops by changing amino acid composition or increasing protein content, reductions in pesticide usage, and herbicide usage by transferring insect, disease and herbicide resistant genes into cultivars. Genetic transformation technologies such as Agrobacterium-mediated and microprojectile bombardment-mediated transformation techniques have really been developed to supplement conventional plant breeding techniques. The introduction and expression of foreign DNA have been used to modify basic aspects of physiology and development and to introduce commercially important characteristics such as herbicide and insect resistance into plants. Agrobacterium-based transformation systems have been widely used in dicotyledonous plant species. Most monocotyledonous plants are insensitive to Agrobacterium infection, and subsequent transformation are not successful. Several techniques for direct DNA delivery into cells including chemical mediated uptake of DNA into isolated protoplasts, electroporation, injection, and the use of high-velocity particles to introduce foreign DNA into intact tissue. However, all of these transformation techniques involve plant cell and tissue culture and subsequent plant regeneration, which sometimes is not easy to achieve because of genotype dependence.
In accordance with the present invention, a direct method for the delivery of foreign DNA into plants is the pollen tube pathway. This method involves applying foreign DNA onto the excised style of a recipient plant after pollination. Although the pollen tube pathway is not widely accepted because molecular evidence to confirm the genetic transformation by this method is limited, this technique has significant potential as a plant genetic transformation system. It has the following advantages over Agrobacterium-mediated or other direct gene delivery systems: (1) elimination of cell or tissue culture and regeneration systems; (2) low cost; (3) genotype independence; (4) avoidance of somaclonal variation; and (5) higher transformation efficiency.
The objective of this research was to (1) optimize the timing of exogenous DNA application after pollination, which was crucial for the transformation of meristematic cells that give rise to reproductive structures; (2) investigate the effect of different components in exogenous DNA solution on the delivery of foreign DNA; and (3) recover whole transgenic onion plants via pollen tube pathway.